The immediate aim of this research is to study the molecular events of cellular transport, in order to achieve the long-range goal of understanding important physiological functions such as intestinal absorption, renal clearance, as well as certain disease states brought about by abnormalities in cellular transport. We are attempting to isolate membrane-bound transport components from various eukaryotic cells such as Ehrlich ascites tumor cells, 3T3 and SV3T3 mouse cells, and Chinese hamster ovary cells. As a first step in this project, we are characterizing the number and kinds of transport systems for the amino acids in these mammalian cells. In addition, membrane vesicle preparations will be made and the ability of these preparations to bind or accumulate amino acids will be measured. The membrane preparations will be measured. The membrane preparations will be solubilized using detergents and phospholipids and subjected to protein separation techniques. Affinity chromatography and double labeling techniques will be used to aid the isolation and identification of the receptor sites for the amino acid transport systems of the animal cells. Since we have already detected binding activity for leucine in membrane preparations from Ehrlich tumor cells, we will attempt to isolate and characterize the receptor site using conventional methods of protein purification. If successful, we plan to attempt to reconstitute the leucine transport system by adding the purified leucine-binding protein back to membranes or membrane phospholipids.